DOCSLIB.ORG

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled, Matthew A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled, Matthew A EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled, Matthew A. Dragovich, Kieran Adam, Marianne Strazza, Anna S. Tocheva, Irving E. Vega and This information is current as Adam Mor of September 26, 2021. J Immunol published online 1 October 2018 http://www.jimmunol.org/content/early/2018/09/28/jimmun ol.1800839 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/09/28/jimmunol.180083 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 1, 2018, doi:10.4049/jimmunol.1800839 The Journal of Immunology EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled,*,† Matthew A. Dragovich,* Kieran Adam,* Marianne Strazza,* Anna S. Tocheva,* Irving E. Vega,‡ and Adam Mor*,x Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell– mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important Downloaded from regulator of T cell cytotoxicity and further studies should evaluate its role in T cell–mediated inflammation. The Journal of Immunology, 2018, 201: 000–000. he immunological synapse (IS) is the interface between Most of the immune checkpoints are coinhibitory receptors that T cells and APC. The IS consists of segregated clusters of directly inhibit signaling downstream of the TCR. T proteins involved in T cell activation. A key member of Programmed cell death 1 (PD-1) is a typical coinhibitory re- http://www.jimmunol.org/ these clusters is the TCR that is engaged by an Ag-loaded MHC ceptor,expressedonactivatedTcellsandclustersintheIS on the APC. Productive interactions initiate signaling cascades alongside the TCR upon engagement by its ligands, PD-1 ligand 1 that result in T cell adhesion, proliferation, and cytokine secretion. (PDL1) or PDL2 (4). PDL1 is widely expressed on both hema- When the IS is composed of a cytotoxic T cell and a corresponding topoietic and nonhematopoietic cells, including tumor cells, target cell (such as a tumor or virally infected cell), lytic granules whereas PDL2 is mainly expressed on APC (5–9). Upon ligation, are released to eliminate the counter cells. To execute these func- PD-1 recruits the phosphatase SHP2 to dephosphorylate phos- tions, a stable synapse between the T cell and the target cell must phoproteins downstream of the TCR and to inhibit T cell acti- be formed. Indeed, TCR-induced synapses are characterized by vation (10–15). PD-1 knockout mice develop inflammatory reorientation of the microtubule-organizing center and substantial cardiomyopathy and glomerulonephritis (16, 17), and PD-1 block- by guest on September 26, 2021 actin accumulation at the boundaries of the contact region (1–3). ade induces potent antitumor immune response in patients with Whereas T cell functions are required for mounting protective various types of malignancies (18, 19). immune responses, it can also result in autoimmunity unless tightly We have recently used affinity purification mass spectrometry regulated. Accordingly, immune responses are restrained by a (MS) to discover proteins, besides SHP2, that interact with the plethora of mechanisms, including thymic clonal deletion of self- intracellular tail of PD-1 and regulate its function (14). Because reactive T cells, inhibitory cells such as regulatory T cells, and anti- recruitment of PD-1 to the IS is critical for its function (4), we inflammatory cytokines (i.e., IL-10 and TGF-b). Another important used this data set to screen for proteins that interact with PD-1 and mechanism to restrict immune responses is immune checkpoints. that also regulate IS formation through cytoskeleton organization. Multiple proteins were uncovered, including Swiprosin-1/EF hand domain family member D2 (EFHD2). EFHD2 knockdown (KD) *Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016; †Pulmonary Department, The Chaim Sheba Medical Center, Ramat Gan blocked PD-1 inhibitory functions in T cells through inhibition of 52620, Israel; ‡Department of Translational Science and Molecular Medicine, Col- stable IS formation. However, EFHD2 was also required for lege of Human Medicine, Michigan State University, Grand Rapids, MI 49503; and x T cell–mediated cytotoxicity and for antitumor immune response, Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY 10032 implying a pervasive role for this protein in IS formation. ORCID: 0000-0003-3106-1183 (M.S.). Received for publication June 18, 2018. Accepted for publication August 24, 2018. Materials and Methods This work was supported by a grant from the National Institutes of Health (R01 General reagents AI25640). RPMI 1640 medium, DMEM, Dulbecco’s PBS, and FBS were purchased Address correspondence and reprint requests to Dr. Adam Mor, 630 W 168 Street, from Life Technologies. Opti-MEM I was purchased from Invitrogen. PH8-406, Columbia University Medical Center, New York, NY 10032. E-mail Ficoll–Paque was purchased from GE Healthcare. Staphylococcus en- address: [email protected] terotoxin E (SEE) was acquired from Toxin Technology. BCA assay was The online version of this article contains supplemental material. purchased from Pierce Biotechnology. Poly-L-lysine, puromycin, and fibro- Abbreviations used in this article: EFHD2, EF hand domain family member D2; IS, nectin were obtained from Sigma-Aldrich. immunological synapse; ITSM, immunoreceptor tyrosine-based switch motif; KD, knockdown; MS, mass spectrometry; MTS, Aqueous One Solution Cell Proliferation; Cell culture, transfection, and stimulation NYU, New York University; PD-1, programmed cell death 1; PDL1, PD-1 ligand 1; SEE, Staphylococcus enterotoxin E; siRNA, small interfering RNA; TIL, tumor- Primary human T cells were isolated from whole blood using RosetteSep infiltrating lymphocyte; 39-UTR, 39-untranslated region; WT, wild type. (STEMCELL Technologies). Cells were maintained in enriched media at 5% CO2 at 37˚C. Primary murine T cells were isolated from spleens of Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 10- to 12-wk-old mice, followed by negative selection using Dynabeads www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800839 2 EFHD2 IS REQUIRED FOR T CELL CYTOTOXICITY (Invitrogen) T cell isolation kit. Jurkat T cells and Raji B cells were Flow cytometry obtained from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS and 100 U/ml peni- T cells were stained with fluorescently conjugated Abs specific for PD-1 in cillin and streptomycin. MC38 cells were provided by Benjamin Neel FACS Buffer, washed, and fixed in 1% paraformaldehyde. Events were (New York University [NYU]) and maintained in RPMI 1640 medium recorded using FACSCalibur (BD Biosciences) and analyzed using FlowJo supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. software. To obtain single-cell suspensions from tumors, tissues were HEK 293T cells were obtained from the American Type Culture Col- digested and cells were blocked with anti-FcR prior to staining with Abs against CD3, PD-1, CD44, CD8, CD4, and CD45 (BioLegend). For anti- lection and maintained in 5% CO2 at 37˚C in DMEM media supple- mented with 10% FBS and 100 U/ml penicillin and streptomycin. DNA Foxp3 (BioLegend) staining, cells were fixed and permeabilized using expression constructs were introduced into the cells by nucleofection True-Nuclear Transcription Factor Buffer Set (BioLegend). Samples were (Lonza Bioscience) with efficiency of 50–70%. Cells were stimulated collected on a BD LSR II Flow Cytometer (BD Biosciences), and data with magnetic beads (ratio of 1:3 cells per bead), which were conjugated were analyzed using FlowJo software. with the following protein combinations (the ratio in parentheses Static adhesion assay indicates the relative concentration of each protein): anti-CD3/IgG1 (1:3), anti-CD3/PDL1-Fc/IgG1 (1:2:1), anti-CD3/PDL2-Fc/IgG1 (1:2:1), Static T cell adhesion to fibronectin-coated plates was performed as anti-CD3/anti-CD28/IgG1 (1:1:2), or anti-CD3/anti-CD28/PDL1-Fc reported earlier (21). (1:1:2). Constructs were introduced into HEK 293T cells using Super- Fect Transfection Reagent (Qiagen) according to the manufacturer’s Conjugate formation assay protocol. SEE (Toxin Technology)-loaded Raji B cells (1.5 3 106) were mixed with 6 Abs Jurkat T cells (1.5 3 10 ), plated on poly-L-lysine (5 mg/ml)–coated glass bottom culture 35-mm plates (ibidi), and subjected to brief centrifugation Anti-CD3 (UCHT1), PDL1-Fc, and PDL2-Fc were purchased from R&D prior to imaging.
Load more

Recommended publications
  • Structure of the Dock2âˆ'elmo1 Complex Provides Insights Into
    ARTICLE https://doi.org/10.1038/s41467-020-17271-9 OPEN Structure of the DOCK2−ELMO1 complex provides insights into regulation of the auto-inhibited state Leifu Chang1,7, Jing Yang1, Chang Hwa Jo 2, Andreas Boland 1,8, Ziguo Zhang 1, Stephen H. McLaughlin 1, Afnan Abu-Thuraia 3, Ryan C. Killoran2, Matthew J. Smith 2,4,9, Jean-Francois Côté 3,5,6,9 & ✉ David Barford 1 DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange 1234567890():,; factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2−ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG−ELMO2RBD complex. The binary DOCK2−ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto- inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF. 1 MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. 2 Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3T 1J4, Canada. 3 Montreal Institute of Clinical Research (IRCM), Montréal, QC H2W 1R7, Canada.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Genetic and Genomic Analysis of Hyperlipidemia, Obesity and Diabetes Using (C57BL/6J × TALLYHO/Jngj) F2 Mice
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Nutrition Publications and Other Works Nutrition 12-19-2010 Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P. Stewart Marshall University Hyoung Y. Kim University of Tennessee - Knoxville, [email protected] Arnold M. Saxton University of Tennessee - Knoxville, [email protected] Jung H. Kim Marshall University Follow this and additional works at: https://trace.tennessee.edu/utk_nutrpubs Part of the Animal Sciences Commons, and the Nutrition Commons Recommended Citation BMC Genomics 2010, 11:713 doi:10.1186/1471-2164-11-713 This Article is brought to you for free and open access by the Nutrition at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Nutrition Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Stewart et al. BMC Genomics 2010, 11:713 http://www.biomedcentral.com/1471-2164/11/713 RESEARCH ARTICLE Open Access Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P Stewart1, Hyoung Yon Kim2, Arnold M Saxton3, Jung Han Kim1* Abstract Background: Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/ JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.
    [Show full text]
  • CD29 Identifies IFN-Γ–Producing Human CD8+ T Cells With
    + CD29 identifies IFN-γ–producing human CD8 T cells with an increased cytotoxic potential Benoît P. Nicoleta,b, Aurélie Guislaina,b, Floris P. J. van Alphenc, Raquel Gomez-Eerlandd, Ton N. M. Schumacherd, Maartje van den Biggelaarc,e, and Monika C. Wolkersa,b,1 aDepartment of Hematopoiesis, Sanquin Research, 1066 CX Amsterdam, The Netherlands; bLandsteiner Laboratory, Oncode Institute, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; cDepartment of Research Facilities, Sanquin Research, 1066 CX Amsterdam, The Netherlands; dDivision of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; and eDepartment of Molecular and Cellular Haemostasis, Sanquin Research, 1066 CX Amsterdam, The Netherlands Edited by Anjana Rao, La Jolla Institute for Allergy and Immunology, La Jolla, CA, and approved February 12, 2020 (received for review August 12, 2019) Cytotoxic CD8+ T cells can effectively kill target cells by producing therefore developed a protocol that allowed for efficient iso- cytokines, chemokines, and granzymes. Expression of these effector lation of RNA and protein from fluorescence-activated cell molecules is however highly divergent, and tools that identify and sorting (FACS)-sorted fixed T cells after intracellular cytokine + preselect CD8 T cells with a cytotoxic expression profile are lacking. staining. With this top-down approach, we performed an un- + Human CD8 T cells can be divided into IFN-γ– and IL-2–producing biased RNA-sequencing (RNA-seq) and mass spectrometry cells. Unbiased transcriptomics and proteomics analysis on cytokine- γ– – + + (MS) analyses on IFN- and IL-2 producing primary human producing fixed CD8 T cells revealed that IL-2 cells produce helper + + + CD8 Tcells.
    [Show full text]
  • Tyr724 Phosphorylation of ELMO1 by Src Is Involved in Cell Spreading and Migration Via Rac1 Activation
    Title Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation Makino, Yoshinori; Tsuda, Masumi; Ohba, Yusuke; Nishihara, Hiroshi; Sawa, Hirofumi; Nagashima, Kazuo; Tanaka, Author(s) Shinya Cell communication and signaling, 13, 35 Citation https://doi.org/10.1186/s12964-015-0113-y Issue Date 2015-07-26 Doc URL http://hdl.handle.net/2115/59828 Rights(URL) http://creativecommons.org/licenses/by/4.0 Type article File Information s12964-015-0113-y.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Makino et al. Cell Communication and Signaling (2015) 13:35 DOI 10.1186/s12964-015-0113-y RESEARCH ARTICLE Open Access Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation Yoshinori Makino1,2, Masumi Tsuda1, Yusuke Ohba3, Hiroshi Nishihara4, Hirofumi Sawa1,5, Kazuo Nagashima1,6 and Shinya Tanaka1,4* Abstract Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays.
    [Show full text]
  • Structural Basis for Mutual Relief of the Rac Guanine Nucleotide Exchange Factor DOCK2 and Its Partner ELMO1 from Their Autoinhibited Forms
    Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms Kyoko Hanawa-Suetsugua, Mutsuko Kukimoto-Niinoa,b, Chiemi Mishima-Tsumagaria, Ryogo Akasakaa, Noboru Ohsawaa, Shun-ichi Sekinea,c, Takuhiro Itoa,c, Naoya Tochioa, Seizo Koshibaa, Takanori Kigawaa,d, Takaho Teradaa,b, Mikako Shirouzua, Akihiko Nishikimib,e,f, Takehito Urunoe, Tomoya Katakaig, Tatsuo Kinashig, Daisuke Kohdah, Yoshinori Fukuib,e,f,1, and Shigeyuki Yokoyamaa,b,c,1 aRIKEN Systems and Structural Biology Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan; bJapan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo 102-0075, Japan; cDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan; dTokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8502, Japan; eDivision of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; fResearch Center for Advanced Immunology, Kyushu University, Fukuoka 812-8582, Japan; gDepartment of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka 570-8506, Japan; and hDivision of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan Edited by John Kuriyan, University of California, Berkeley, CA, and approved December 31, 2011 (received for review September 6, 2011) DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide for the Rho-family GTPases. There are 11 mammalian members exchange factor, controls lymphocyte migration through ras-related (DOCK180, DOCK2-11) of the CDM family. The CDM proteins C3 botulinum toxin substrate (Rac) activation.
    [Show full text]
  • Fraternal Twins: Swiprosin-1/Efhd2 and Swiprosin-2/Efhd1, Two
    Dütting et al. Cell Communication and Signaling 2011, 9:2 http://www.biosignaling.com/content/9/1/2 REVIEW Open Access Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions Sebastian Dütting, Sebastian Brachs, Dirk Mielenz* Abstract Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present in Bilateria, for instance in Drosophila melanogaster and Caenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands.
    [Show full text]
  • Identification of Two Signaling Submodules Within the Crkii/ELMO
    Cell Death and Differentiation (2007) 14, 963–972 & 2007 Nature Publishing Group All rights reserved 1350-9047/07 $30.00 www.nature.com/cdd Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells A-C Tosello-Trampont1,4, JM Kinchen1,2,4, E Brugnera1,3, LB Haney1, MO Hengartner2 and KS Ravichandran*,1 Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles.
    [Show full text]
  • Gene Polymorphism in Development of Diabetic Kidney Disease Thoria A
    Omar et al. Egyptian Journal of Medical Human Genetics (2021) 22:49 Egyptian Journal of Medical https://doi.org/10.1186/s43042-021-00167-8 Human Genetics RESEARCH Open Access Role of engulfment and cell motility 1 (ELMO1) gene polymorphism in development of diabetic kidney disease Thoria A. Omar1*, Shimaa K. Zewain2, Mohamed M. Ghonaim3, Khadija A. Refaat1 and Dalia H. Abou-Elela1 Abstract Background: Diabetic kidney disease (DKD) is a progressive kidney disease that affects diabetic patients irrespective of glycemic state or hypertension. Therefore, early detection of DKD is of critical importance. Many genome-wide association studies have identified the engulfment and cell motility 1 (ELMO1) gene as a genetic marker linked to DKD. This study aimed to investigate the association between ELMO1 rs741301 gene polymorphism and the development of DKD among Egyptian patients with type 2 diabetes mellitus (T2DM). Allele and genotype frequencies were investigated in 304 subjects by real-time PCR allelic discrimination assay: 100 DKD patients, 102 diabetic patients without DKD, and 102 healthy controls. Results: GG genotype of ELMO1 (rs741301) SNP and its allele frequencies were significantly high in all diabetic patients. GG genotype had an odds ratio (OR) of 6.095 and 95% confidence interval (CI) of 2.456–15.125, p < 0.001, while the frequent allele G had an OR of 2.366 and 95% CI of 1.450–3.859, p = 0.001. No significant difference was observed between T2DM without DKD and DKD. Conclusion: Our results could not establish an association between the ELMO1 rs741301 variant and the progression of DKD.
    [Show full text]
  • ELMO1 Signaling Is a Promoter of Osteoclast Function and Bone Loss ✉ Sanja Arandjelovic 1 , Justin S
    ARTICLE https://doi.org/10.1038/s41467-021-25239-6 OPEN ELMO1 signaling is a promoter of osteoclast function and bone loss ✉ Sanja Arandjelovic 1 , Justin S. A. Perry 1, Ming Zhou 2, Adam Ceroi3, Igor Smirnov4, Scott F. Walk1, Laura S. Shankman1, Isabelle Cambré3, Suna Onengut-Gumuscu 5, Dirk Elewaut 3, Thomas P. Conrads 2 & ✉ Kodi S. Ravichandran 1,3 Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. 1234567890():,; Here, we identify the cytoplasmic protein ELMO1 as an important ‘signaling node’ in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the ‘ELMO1 inter- actome’ in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis. 1 Center for Cell Clearance, Department of Microbiology, Immunology, and Cancer Biology and Carter Immunology Center, University of Virginia, Charlottesville, VA, USA. 2 Inova Schar Cancer Institute, Inova Center for Personalized Health, Fairfax, VA, USA. 3 Inflammation Research Centre, VIB, and the Department of Biomedical Molecular Biology, Ghent Univeristy, Ghent, Belgium.
    [Show full text]
  • POGLUT1, the Putative Effector Gene Driven by Rs2293370 in Primary
    www.nature.com/scientificreports OPEN POGLUT1, the putative efector gene driven by rs2293370 in primary biliary cholangitis susceptibility Received: 6 June 2018 Accepted: 13 November 2018 locus chromosome 3q13.33 Published: xx xx xxxx Yuki Hitomi 1, Kazuko Ueno2,3, Yosuke Kawai1, Nao Nishida4, Kaname Kojima2,3, Minae Kawashima5, Yoshihiro Aiba6, Hitomi Nakamura6, Hiroshi Kouno7, Hirotaka Kouno7, Hajime Ohta7, Kazuhiro Sugi7, Toshiki Nikami7, Tsutomu Yamashita7, Shinji Katsushima 7, Toshiki Komeda7, Keisuke Ario7, Atsushi Naganuma7, Masaaki Shimada7, Noboru Hirashima7, Kaname Yoshizawa7, Fujio Makita7, Kiyoshi Furuta7, Masahiro Kikuchi7, Noriaki Naeshiro7, Hironao Takahashi7, Yutaka Mano7, Haruhiro Yamashita7, Kouki Matsushita7, Seiji Tsunematsu7, Iwao Yabuuchi7, Hideo Nishimura7, Yusuke Shimada7, Kazuhiko Yamauchi7, Tatsuji Komatsu7, Rie Sugimoto7, Hironori Sakai7, Eiji Mita7, Masaharu Koda7, Yoko Nakamura7, Hiroshi Kamitsukasa7, Takeaki Sato7, Makoto Nakamuta7, Naohiko Masaki 7, Hajime Takikawa8, Atsushi Tanaka 8, Hiromasa Ohira9, Mikio Zeniya10, Masanori Abe11, Shuichi Kaneko12, Masao Honda12, Kuniaki Arai12, Teruko Arinaga-Hino13, Etsuko Hashimoto14, Makiko Taniai14, Takeji Umemura 15, Satoru Joshita 15, Kazuhiko Nakao16, Tatsuki Ichikawa16, Hidetaka Shibata16, Akinobu Takaki17, Satoshi Yamagiwa18, Masataka Seike19, Shotaro Sakisaka20, Yasuaki Takeyama 20, Masaru Harada21, Michio Senju21, Osamu Yokosuka22, Tatsuo Kanda 22, Yoshiyuki Ueno 23, Hirotoshi Ebinuma24, Takashi Himoto25, Kazumoto Murata4, Shinji Shimoda26, Shinya Nagaoka6, Seigo Abiru6, Atsumasa Komori6,27, Kiyoshi Migita6,27, Masahiro Ito6,27, Hiroshi Yatsuhashi6,27, Yoshihiko Maehara28, Shinji Uemoto29, Norihiro Kokudo30, Masao Nagasaki2,3,31, Katsushi Tokunaga1 & Minoru Nakamura6,7,27,32 Primary biliary cholangitis (PBC) is a chronic and cholestatic autoimmune liver disease caused by the destruction of intrahepatic small bile ducts. Our previous genome-wide association study (GWAS) identifed six susceptibility loci for PBC.
    [Show full text]
  • Systematic Elucidation of Neuron-Astrocyte Interaction in Models of Amyotrophic Lateral Sclerosis Using Multi-Modal Integrated Bioinformatics Workflow
    ARTICLE https://doi.org/10.1038/s41467-020-19177-y OPEN Systematic elucidation of neuron-astrocyte interaction in models of amyotrophic lateral sclerosis using multi-modal integrated bioinformatics workflow Vartika Mishra et al.# 1234567890():,; Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Net- works (SEARCHIN) to identify ligand-mediated interactions between distinct cellular com- partments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication
    [Show full text]